Glaucoma is a progressive optic neuropathy characterized by degeneration of retinal ganglion cells (RGC) and loss of visual field. After cataracts, its the second leading cause of blindness worldwide. Glaucoma has a multifactorial etiology and the mechanisms leading RGC loss are not yet fully clear. Increased intraocular pressure (IOP) represents the main risk factor and, currently, the only modifiable factor by commercial drugs. To date, avaiable therapeutic approaches can only slow down the progression of the disease and do not allow to stop it. In this context, one of the major goals of glaucoma research is elucidating the mechanisms leading to RGCs death. This thesis is based on the hypothesis that the RGC neurodegeneration originates from an imbalance between damaging factors and endogenous neuroprotective factors. HuR/ELAV1 protein plays a neuroprotective role through the post-transcriptional regulation of genes encoding proteins involved in cellular stress response. The aim of this thesis is the evaluation of expression and subcellular distribution alterations of HuR protein in the retina and optic nerve of glaucomatous rats, on HuR target expression. In particular, experiments were performed on retinas and optic nerves from in vivo glaucoma model in which IOP increase was obtained by the iniection of polystyrene microbeads into the anterior chamber of the eye that leads to obstruction of the aqueous humor outflow pathways. In addition to animal studies, human retinal samples from healthy donors and patients with primary open-angle glaucoma were also analyzed. The results reveal that both human and animal samples - the latter exposed to elevated IOP for 8 weeks -, display an alteration in HuR function and, specifically, an impairment of its protein level and changes in its nuclear-cytoplasmic shuttling. The alteration of HuR post-transcriptional control leads to decreased gene expression of proteins crucial in mounting adequate RGCs stress response, such as Hsp70, and an imbalance in the homeostasis of HuR-target RNAs. The increase of p53 in the retina is a possible consequence of this imbalance and likely the trigger of RGC death detectable in glaucomatous rats. These results are the first in vivo evidence of a potential involvement of HuR in glaucoma.
Il glaucoma è una neuropatia ottica progressiva caratterizzata dalla degenerazione delle cellule gangliari retiniche (RGC) con conseguente perdita del campo visivo. A livello mondiale rappresenta la seconda causa di cecità dopo la cataratta. Si tratta di una patologia ad eziologia multifattoriale in cui i meccanismi che portano alla perdita delle RGC non sono ancora del tutto chiari. Un aumento della pressione intraoculare (IOP) rappresenta il fattore di rischio principale e lunico attualmente modificabile. Ad oggi, sono disponibili approcci terapeutici che rallentano la progressione della malattia, ma non consentono di arrestarla. In questo contesto, molte ricerche sul glaucoma sono volte a comprendere i meccanismi che conducono alla perdita delle RGC. Tale lavoro di tesi si fonda sullipotesi secondo cui la neurodegenerazione delle RGC origina da uno sbilanciamento dellequilibrio tra fattori di danno e fattori neuroprotettivi endogeni. La proteina HuR/ELAV1 svolge un ruolo neuroprotettivo attraverso la regolazione post-trascrizionale di geni che codificano per proteine coinvolte nello sviluppo della risposta cellulare allo stress. Oggetto specifico di questa tesi è lo studio dellalterazione dellespressione e della distribuzione subcellulare di HuR nella retina e nel nervo ottico in un modello animale di glaucoma ad angolo aperto in cui la IOP è stata aumentata attraverso liniezione di microbeads di polistirene nella camera anteriore dellocchio. Inoltre, sono stati analizzati anche campioni umani di retina provenienti da donatori sani e affetti da glaucoma primario ad angolo aperto. I risultati rivelano che, in seguito a glaucoma sia nei tessuti sia nei campioni umani, si verifica unalterazione di HuR/ELAVL1. In particolare, si riscontrano ridotti livelli proteici e unalterata distribuzione tra nucleo e citoplasma che suggeriscono una sua ridotta funzionalità e una compromissione nellequilibrio tra i trascritti target di HuR. Questo si traduce, nelle RGC, in una diminuita espressione di proteine fondamentali per lo sviluppo di unadeguata risposta cellulare allo stress, come Hsp70, e un aumento di p53 verosimilmente responsabile dellinduzione dellapoptosi, osservabile nelle RGC degli animali glaucomatosi. Questi dati rappresentano la prima evidenza in vivo del coinvolgimento di HuR nella patogenesi del glaucoma e di un suo potenziale come nuovo target terapeutico per la prevenzione della perdita delle RGC.
Proteina HuR/ELAVL1 e glaucoma: studio in vivo di un potenziale nuovo target terapeutico
ROSSI, CECILIA
2018/2019
Abstract
Glaucoma is a progressive optic neuropathy characterized by degeneration of retinal ganglion cells (RGC) and loss of visual field. After cataracts, its the second leading cause of blindness worldwide. Glaucoma has a multifactorial etiology and the mechanisms leading RGC loss are not yet fully clear. Increased intraocular pressure (IOP) represents the main risk factor and, currently, the only modifiable factor by commercial drugs. To date, avaiable therapeutic approaches can only slow down the progression of the disease and do not allow to stop it. In this context, one of the major goals of glaucoma research is elucidating the mechanisms leading to RGCs death. This thesis is based on the hypothesis that the RGC neurodegeneration originates from an imbalance between damaging factors and endogenous neuroprotective factors. HuR/ELAV1 protein plays a neuroprotective role through the post-transcriptional regulation of genes encoding proteins involved in cellular stress response. The aim of this thesis is the evaluation of expression and subcellular distribution alterations of HuR protein in the retina and optic nerve of glaucomatous rats, on HuR target expression. In particular, experiments were performed on retinas and optic nerves from in vivo glaucoma model in which IOP increase was obtained by the iniection of polystyrene microbeads into the anterior chamber of the eye that leads to obstruction of the aqueous humor outflow pathways. In addition to animal studies, human retinal samples from healthy donors and patients with primary open-angle glaucoma were also analyzed. The results reveal that both human and animal samples - the latter exposed to elevated IOP for 8 weeks -, display an alteration in HuR function and, specifically, an impairment of its protein level and changes in its nuclear-cytoplasmic shuttling. The alteration of HuR post-transcriptional control leads to decreased gene expression of proteins crucial in mounting adequate RGCs stress response, such as Hsp70, and an imbalance in the homeostasis of HuR-target RNAs. The increase of p53 in the retina is a possible consequence of this imbalance and likely the trigger of RGC death detectable in glaucomatous rats. These results are the first in vivo evidence of a potential involvement of HuR in glaucoma.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/25029